Fusion proteins of humanized g250 specific antibodies and uses thereof

ABSTRACT

Chimeric antibodies, as well as fusion proteins which comprise chimeric antibodies, are disclosed. The antibodies bind to GM-CSF, CD-30, and G250 antigen. The fusion proteins include biologically active portions of tumor necrosis factor, or full length tumor necrosis factor. Expression vectors adapted for production of the antibodies, as well as methods for manufacturing these, are also disclosed.

FIELD OF THE INVENTION

This invention relates to the field of molecular immunology, generally,and to vectors useful for expression of proteins, especially antibodies,such as fully human, humanized, and chimeric antibodies, as well asfusion proteins which incorporate the antibody and a protein or proteinfragment, in eukaryotic cells, mammalian cells in particular. Theresulting antibodies and fusion proteins are also a feature of theinvention.

BACKGROUND AND PRIOR ART

One serious problem with using murine antibodies for therapeuticapplications in humans is that they quickly raise a human anti-mouseresponse (HAMA) which reduces the efficacy of the antibody in patients,and prevents continued administration thereof. Parallel issues arisewith the administration of antibodies from other, non-human species. Oneapproach to overcoming this problem is to generate so-called “chimeric”antibodies. These can comprise murine variable regions, and humanconstant regions (Boulianne et al. (1984) Nature 312(5995): 643-646.;incorporated by reference herein in its entirety). Although chimericantibodies contain murine sequences and can elicit an anti-mouseresponse in humans (LoBuglio et al. (1989) Proc. Natl. Acad. Sci. USA86(11): 4220-4224; incorporated by reference herein in its entirety),trials with chimeric antibodies in the area of hematological disease(e.g., Non-Hodgkin-Lymphoma; Witzig et al. (1999) J. Clin. Oncol.17(12): 3793-3803.; incorporated by reference herein in its entirety) orautoimmune disease (e.g., rheumatoid arthritis, chronic inflammatorybowel disease; Van den Bosch; et al, Lancet 356(9244):1821-2 (2000),incorporated by reference herein in its entirety) have led to FDAapproval and demonstrate that these molecules have significant clinicalpotential and efficacy.

Recent studies have indicated that granulocyte-macrophage colonystimulating growth factor (GM-CSF) plays a role in the development ofrheumatoid arthritis (RA) (Cook, et al., Arthritis Res. 2001, 3:293-298,incorporated by reference herein in its entirety) and possibly otherinflammatory diseases and conditions. Therefore, it would be of interestto develop a drug which would block GM-CSF and its effect on cells. Thepresent invention provides a chimeric antibody, targeting the GM-CSFmolecule, which has blocking capacity.

The increased use of chimeric antibodies in therapeutic applications hascreated the need for expression vectors that effectively and efficientlyproduce high yields of functional chimeric antibodies in eukaryoticcells, such as mammalian cells, which are preferred for production. Thepresent invention provides novel expression vectors, transformed hostcells and methods for producing chimeric antibodies in mammalian cells,as well as the antibodies themselves and fusion proteins containingthem.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the binding of recombinant, chimeric anti GM-CSF antibodyvia Western Blotting.

FIG. 2 shows the binding of the antibody via ELISA.

FIG. 3 shows the blocking effect of the antibody on GM-CSF growthdependent TF-1 cells.

FIG. 4 shows the blocking effect of the antibody on GM-CSF growthdependent AML-193 cells.

FIG. 5 shows results of an assay testing the effect of increasingconcentration of murine or chimeric 19/2 mAbs, on TF-1 cells grown inthe presence of a constant amount of human GM-CSF.

FIG. 6 parallels the experiment of FIG. 5, but uses the AML-153 cells.

FIG. 7 shows a schematic map of the two expression vectors used toprepare the recombinant antibodies.

SUMMARY OF INVENTION

The present invention provides expression vectors which are useful inthe expression of proteins, such as antibodies, especially fully human,humanized or chimerized antibodies, and fusion proteins containingthese. Both light chains and heavy chains can be expressed. Theexpression vectors of the present invention comprise a human elongationfactor 1 α (EF1α) promoter/enhancer sequence, an internal ribosome entrysite (IRES) sequence (U.S. Pat. No. 4,937,190; incorporated herein inits entirety), a nucleotide sequence that confers neomycin resistance toa cell containing the expression vector, and a nucleotide sequence undercontrol of a simian virus 40 promoter (SV40) that confers ampicillinresistance to a cell containing the expression vector. In a preferredembodiment, the EF1α promoter/enhancer sequence is upstream and adjacentto a nucleotide sequence encoding a chimeric light chain.

The expression vector of the present invention may contain a nucleotidesequence encoding any immunoglobulin light chain. In a preferredembodiment the light chain variable region is of murine origin, and thelight chain constant region is either human kappa or human lambda. In amore preferred embodiment, the chimeric light chain variable region isderived from a murine antibody that binds to GM-CSF, CD-30, or G250 andin especially preferred embodiments, to the human forms of thesemolecules.

The present invention also provides a further expression vector usefulin the expression of proteins, such as antibodies, especially fullyhuman, humanized or chimeric antibodies, and fusion proteins containingthese. This second embodiment differs from the first in that instead ofthe neomycin resistance sequence, described supra, it comprises anucleotide sequence which encodes dihydrofolate reductase or “dhfr,”which generates resistance against the well known selection markermethotrexate. Such an expression vector may contain nucleotide sequencesencoding any antibody or portion thereof, such as heavy or light chainsof fully human, humanized or chimerized antibodies. In a preferredembodiment, a heavy chain is expressed, where the variable region is ofmurine origin, and the heavy chain constant region is human IgG1. In amore preferred embodiment, the chimeric heavy chain variable region isderived from a murine antibody that binds CD-30, GM-CSF or G250,preferably the human forms of these.

In another embodiment, the present invention provides host cellstransformed or transfected with any one of the expression vectors of thepresent invention. In a preferred embodiment, a host cell, preferably aeukaryotic cell, more preferably a mammalian cell, is transformed ortransfected with an expression vector comprising a chimericimmunoglobulin light chain and an expression vector comprising achimeric immunoglobulin heavy chain. The present invention contemplatesprokaryotic and eukaryotic cells, such as mammalian cells, insect cells,bacterial or fungal cells. In a preferred embodiment, the host cell is ahuman or Chinese Hamster Ovary (“CHO”) cell.

The present invention also provides methods for the recombinantproduction of a chimeric immunoglobulin light or heavy chain comprisingthe step of culturing a transformed or transfected host cell of thepresent invention. In one embodiment, the methods of the presentinvention further comprise the isolation of the chimeric immunoglobulinlight or heavy chain.

The present invention also provides methods for the recombinantproduction of a fully human, humanized or chimeric immunoglobulincomprising culturing a host cell that has been transformed ortransfected with an expression vector comprising a chimericimmunoglobulin light chain and an expression vector comprising achimeric immunoglobulin heavy chain, or an expression vector encodesboth chains. In one embodiment, the methods of the present inventionfurther comprise the self-assembly of the chimeric heavy and light chainimmunoglobulins and isolation of the chimeric immunoglobulin. Methodsfor accomplishing this are well known in the art.

The present invention also provides the chimeric immunoglobulin lightchain, heavy chain or assembled chimeric immunoglobulin produced by themethods of the present invention. In another embodiment, the presentinvention provides compositions comprising the chimeric immunoglobulinlight chain, heavy chain or assembled chimeric immunoglobulin of thepresent invention and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF INVENTION

1. Definitions

As used herein “chimerized” refers to an immunoglobulin such as anantibody, wherein the heavy and light chains of the variable regions arenot of human origin and wherein the constant regions of the heavy andlight chains are of human origin.

“Humanized” refers to an immunoglobulin such as an antibody, wherein theamino acids directly involved in antigen binding, the so-calledcomplementary determining regions (CDR), of the heavy and light chainsare not of human origin, while the rest of the immunoglobulin molecule,the so-called framework regions of the variable heavy and light chains,and the constant regions of the heavy and light chains are of humanorigin.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Immunoglobulin” or “antibody” refers to any member of a group ofglycoproteins occurring in higher mammals that are major components ofthe immune system. As used herein, “immunoglobulins” and “antibodies”comprise four polypeptide chains-two identical light chains and twoidentical heavy chains that are linked together by disulfide bonds. Animmunoglobulin molecule includes antigen binding domains, which eachinclude the light chains and the end-terminal portion of the heavychain, and the Fc region, which is necessary for a variety of functions,such as complement fixation. There are five classes of immunoglobulinswherein the primary structure of the heavy chain, in the Fc region,determines the immunoglobulin class. Specifically, the alpha, delta,epsilon, gamma, and mu chains correspond to IgA, IgD, IgE, IgG and IgM,respectively. As used herein “immunoglobulin” or “antibody” includes allsubclasses of alpha, delta, epsilon, gamma, and mu and also refers toany natural (e.g., IgA and IgM) or synthetic multimers of the four-chainimmunoglobulin structure.

“Antigen-binding fragment”, “antigen-binding domain” and “Fab fragment”all refer to the about 45 kDa fragment obtained by papain digestion ofan immunoglobulin molecule and consists of one intact light chain linkedby a disulfide bond to the N-terminal portion of the contiguous heavychain. As used herein, “F(ab)₂ fragment” refers to the about 90 kDaprotein produced by pepsin hydrolysis of an immunoglobulin molecule. Itconsists of the N-terminal pepsin cleavage product and contains bothantigen binding fragments of a divalent immunoglobulin, such as IgD,IgE, and IgG. Neither the “antigen-binding fragment” nor “F(ab)₂fragment” contain the about 50 kDa F_(c) fragment produced by papaindigestion of an immunoglobulin molecule that contains the C-terminalhalves of the immunoglobulin heavy chains, which are linked by twodisulfide bonds, and contain sites necessary for compliment fixation.

“Epitope” refers to an immunological determinant of an antigen thatserves as an antibody-binding site. Epitopes can be structural orconformational.

“Hybridoma” refers to the product of a cell-fusion between a culturedneoplastic lymphocyte and a normal, primed B- or T-lymphocyte, whichexpresses the specific immune potential of the parent cell.

“Heavy chain” refers to the longer & heavier of the two types ofpolypeptide chain in immunoglobulin molecules that contain the antigenicdeterminants that differentiate the various Ig classes, e.g., IgA, IgD,IgE, IgG, IgM, and the domains necessary for complement fixation,placental transfer, mucosal secretion, and interaction with F_(c)receptors.

“Light chain” refers to the shorter & lighter of the two types ofpolypeptide chain in an Ig molecule of any class. Light chains, likeheavy chains, comprise variable and constant regions.

“Heavy chain variable region” refers to the amino-terminal domain of theheavy chain that is involved in antigen binding and combines with thelight chain variable region to form the antigen-binding domain of theimmunoglobulin.

“Heavy chain constant region” refers to one of the three heavy chaindomains that are carboxy-terminal portions of the heavy chain.

“Light chain variable region” refers to the amino-terminal domain of thelight chain and is involved in antigen binding and combines with theheavy chain to form the antigen-binding region.

“Light chain constant region” refers to the one constant domain of eachlight chain. The light chain constant region consists of either kappa orlambda chains.

“Murine anti-human-GM-CSF 19/2 antibody” refers to a murine monoclonalantibody that is specific for human GM-CSF. This antibody is well knownand it has been studied in detail. See Dempsey, et al, Hybridoma9:545-58 (1990); Nice, et al, Growth Factors 3:159-169 (1990), bothincorporated by reference.

“Effective amount” refers to an amount necessary to produce a desiredeffect.

“Antibody” refers to any glycoprotein of the immunoglobulin family thatnon-covalently, specifically, and reversibly binds a correspondingantigen.

“Monoclonal antibody” refers to an immunoglobulin produced by a singleclone of antibody-producing cells. Unlike polyclonal antiserum,monoclonal antibodies are monospecific (e.g., specific for a singleepitope of a single antigen).

“Granulocytes” include neutrophils, eosinophils, and basophils.

“GM-CSF” refers to a family of glycoprotein growth factors that controlthe production, differentiation, and function of granulocytes andmonocytes-macrophages. Exemplary, but by no means the only form of suchmolecules, can be seen in U.S. Pat. No. 5,602,007, incorporated byreference.

“Inflammatory condition” refers to immune reactions that are eitherspecific or non-specific. For example, a specific reaction is an immunereaction to an antigen. Examples of specific reactions include antibodyresponses to antigens, such as viruses and allergens, includingdelayed-type hypersensitivity, including psoriasis, asthma, delayed typehypersensitivity, inflammatory bowel disease, multiple sclerosis, viralpneumonia, bacterial pneumonia, and the like. A non-specific reaction isan inflammatory response that is mediated by leukocytes such asmacrophages, eosinophils and neutrophils. Examples of non-specificreactions include the immediate swelling after a bee sting, and thecollection of polymorphonuclear (PMN) leukocytes at sites of bacterialinfection. Other “inflammatory conditions” within the scope of thisinvention include, e.g., autoimmune disorders such as psoriasis,rheumatoid arthritis, lupus, post-ischemic leukocyte mediated tissuedamage (reperfusion injury), frost-bite injury or shock, acuteleukocyte-mediated lung injury (acute respiratory distress syndrome orARDS), asthma, traumatic shock, septic shock, nephritis, acute andchronic inflammation, and platelet-mediated pathologies such asateriosclerosis and inappropriate blood clotting.

“Pharmaceutically acceptable carrier” refers to any carrier, solvent,diluent, vehicle, excipient, adjuvant, additive, preservative, and thelike, including any combination thereof, that is routinely used in theart.

Physiological saline solution, for example, is a preferred carrier, butother pharmaceutically acceptable carriers are also contemplated by thepresent invention. The primary solvent in such a carrier may be eitheraqueous or non-aqueous. The carrier may contain other pharmaceuticallyacceptable excipients for modifying or maintaining pH, osmolarity,viscosity, clarity, color, sterility, stability, rate of dissolution,and/or odor. Similarly, the carrier may contain still otherpharmaceutically acceptable excipients for modifying or maintaining thestability, rate of dissolution, release, or absorption or penetrationacross the blood-brain barrier.

The fully human, humanized or chimerized antibodies of the presentinvention may be administered orally, topically, parenterally, rectallyor by inhalation spray in dosage unit formulations that containconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles. As used herein, “parenterally” refers to subcutaneous,intravenous, intramuscular, intrastemal, intrathecal, and intracerebralinjection, including infusion techniques.

The fully human, humanized or chimerized antibodies may be administeredparenterally in a sterile medium. The antibodies, depending on thevehicle and concentration used, may be suspended or dissolved in thevehicle. Advantageously, adjuvants such as local anesthetics,preservatives and buffering agents can be dissolved in the vehicle. Themost preferred routes of administration of the pharmaceuticalcompositions of the invention are subcutaneous, intramuscular,intrathecal or intracerebral administration. Other embodiments of thepresent invention encompass administration of the composition incombination with one or more agents that are usually and customarilyused to formulate dosages for parenteral administration in either unitdose or multi-dose form, or for direct infusion.

Active ingredient may be combined with the carrier materials in amountsnecessary to produce single dosage forms. The amount of the activeingredient will vary, depending upon the type of antibody used, the hosttreated, the particular mode of administration, and the condition fromwhich the subject suffers. Preferably, the amount of fully human,humanized or chimerized anti-GM-CSF immunoglobulin, for example, is atherapeutically effective amount which is sufficient to decrease aninflammatory response or ameliorate the symptoms of an inflammatorycondition. It will be understood by those skilled in the art, however,that specific dosage levels for specific patients will depend upon avariety of factors, including the activity of the specificimmunoglobulins utilized, the age, body weight, general health, sex,diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy. Administration of the fully human, humanized orchimerized immunoglobulins of the present invention may require eitherone or multiple dosings.

Regardless of the manner of administration, however, the specific doseis calculated according to approximate body weight or body surface areaof the patient. Further refinement of the dosing calculations necessaryto optimize dosing for each of the contemplated formulations isroutinely conducted by those of ordinary skill in the art without undueexperimentation, especially in view of the dosage information and assaysdisclosed herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

EXAMPLES Example 1 Cloning Strategy for 19/2 Heavy (H) and Light (L)Variable (V)-Region Genes

Total RNA from the hybridoma producing murine 19/2 antibody was obtainedby standard RNA isolation techniques (Chomczynski et al. (1987) Anal.Biochem. 162: 156-159.; incorporated by reference herein in itsentirety). First strand cDNA was prepared using a commerciallyavailable, first strand cDNA synthesis kit and priming with d(T) 18 forboth the heavy and light chains (Renner et al. (1998) Biotechniques24(5): 720-722.; incorporated by reference herein in its entirety). Theresulting cDNA was subjected to PCR using combinations of primers forthe heavy and light chains. The nucleotide sequences of the 5′ primersfor the heavy and light chains are shown in Tables 1 and 2 respectively.The 3′ primers are shown in Table 3. The light chain primer hybridizedwithin the mouse kappa constant region not far from the V-C junction.The heavy chain 3′ primer hybridised within the CH-1 constant region ofmouse heavy chain subgroup I not far from the V-CH1 junction.

TABLE 1 Oligonucleotide primers for the 5′ region of Mouse HeavyVariable (MHV) domains. SEQ ID NO: 1 MHV-1:5′ATGAAATGCAGCTGGGTCATSTTCTTC 3′ 1 MHV-2: 5′ATGGGATGGAGCTRATCATSYTCTT 3′2 MHV-3: 5′ATGAAGWTGTGGTTAAACTGGGTTTTT 3′ 3 MHV-4:5′ATGRACTTTGWYTCAGCTTGRTTT 3′ 4 MHV-5: 5′ATGGACTCCAGGCTCAAMAGTTTTCCTT 3′5 MHV-6: 5′ATGGCTGTCYTRGSGCTRCTCTTCTGC 3′ 6 MHV-7:5′ATGGRATGGAGCKGGRTCTTMTCTT 3′ 7 MHV-8: 5′ATGAGAGTGCTGATTCTTTTGTG 3′ 8MHV-9: 5′ATGGMTTGGGTGTGGAMCTTGCTATTCCTG 3′ 9 MHV-10:5′ATGGGCAGACTTACATTCTCATTCCTG 3′ 10 MHV-11:5′ATGGATTTTGGGCTGATTTTTTTTATTG 3′ 11 MHV-12:5′ATGATGGTGTTAAGTCTTCTGTACCTG 3′ 12 NB KEY R = A/G, Y = T/C, W = A/T, K= T/G, M = A/C, S = C/G.

TABLE 2 Oligonucleotides primers for the 5′ region of Mouse KappaVariable (MKV) domains. SEQ ID NO: 1 MKV-1:5′ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG 3′ 13 MKV-2:5′ATGGAGWCAGACACACTCCTGYTATGGGT 3′ 14 MKV-3:5′ATGAGTGTGCTCACTCAGGTCCTGGSGTTG 3′ 15 MKV-4:5′ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG 16 3′ MKV-5:5′ATGGATTTWCAGGTGCAGATTWTCAGCTTC 3′ 17 MKV-6:5′ATGAGGTKCYYTGYTSAGYTYCTGRGG 3′ 18 MKV-7:5′ATGGGCWTCAAGATGGAGTCACAKWYYCWGG 3′ 19 MKV-8:5′ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG 3′ 20 MKV-9:5′ATGGTRTCCWCASCTCAGTTCCTTG 3′ 21 MKV-10: 5′ATGTATATATGTTTGTTGTCTATTTCT3′ 22 MKV-11: 5′ATGGAAGCCCCAGCTCAGCTTCTCTTCC 3′ 23 MKV-12:5′ATGAAGTTTCCTTCTCAACTTCTGCTC 3′ 24 NB KEY R = A/G, Y = T/C, W = A/T, K= T/G, M = A/C, S = C/G.

TABLE 3 Oligonucleotide primers for the 3′ ends of mouse VH and VLgenes. Light chain (MKC): 5′TGGATGGTGGGAAGATG 3′ 25 Heavy chain (MHG):5′CCAGTGGATAGACAGATG 3′ 26

Example 2 Ig Sequences Cloned from the 19/2 Murine Hybridoma

Using the cloning strategy described, supra, PCR products for VH and VLof murine 19/2 were cloned using a commercially available product, andart recognized techniques. For the murine 19/2 VL region, PCR productswere obtained using the mouse kappa constant region primer and primersMKV2 and MKV7 (SEQ ID NOS: 14 & 19). For the mouse 19/2 VH region, PCRproducts were obtained using the mouse gamma 1 constant region primerand primers MHV2, MHV5 and MHV7 (SEQ ID NOS: 2, 5 and 7). Extensive DNAsequencing of the cloned V-region inserts revealed two different lightchain sequences and 2 different heavy chain sequences. Pseudogenes forheavy and light chain were amplified and were eliminated by standardsequence analyses. A novel immunoglobulin-coding sequence was determinedfor both the heavy and light chains. This is set forth at SEQ ID NOS:27, 28, 29 & 30, which present the cDNA and amino acid sequences for themurine 19/2 heavy chain variable region (27 & 28), and the light chainvariable region (29 & 30).

Example 3 Mouse 19/2 Heavy Chain Leader Sequence

When comparing the DNA sequence of the leader sequence for 19/2 heavychain obtained with the primers described supra, with the database, itappeared that the 19/2 HC leader sequence is short (17 amino acids) andunique vis a vis public data bases. Specifically, amino acids 2, 3 and 5were E, L & M, as compared to S, W & F in the data bases. As compared tothe database, hydrophilic amino acids in the N-terminal region wereseparated by neutral or basic ones, respectively; however, since theinfluence of these changes on the secretory capability of the leadersequence is unclear, this sequence was unaltered in further experiments.

Example 4 Construction of Mouse-human Chimeric Genes

The chimeric 19/2 antibody was designed to have the mouse 19/2 VL and VHregions linked to human kappa and gamma-1 constant regions,respectively. PCR primers were used to modify the 5′- and 3′- sequencesflanking the cDNA sequences coding for the mouse 19/2 VL and VH regions.PCR primers specific for 19/2 light chain V-region were designed usingthe sequence of the 19/2 light chain V-region gene obtained. Theseadapted mouse 19/2 variable regions were then subcloned into mammaliancell expression vectors already containing the human kappa (pREN-Neovector) or the gamma-1 (pREN-DHFR vector) constant regions. The vectorsemploy parts of the human elongation factor 1α (EF 1α) promoter/enhancersequence to efficiently transcribe the light and heavy chains. Thevectors also contain an IRES sequence following the multiple cloningsite to allow for stringent, bicistronic expression and control of theindividual selection marker in CHO cells. This pair of vectors was usedin all of the recombinant work described herein, i.e., to manufactureall chimeric antibodies. The expression vectors were designed to havethe variable regions inserted as PmeI-BamHI DNA fragments. PCR primerswere designed to introduce these restrictions sites at the 5′- (PmeI)and 3′- (BamHI) ends of the cDNAs coding for the V-regions. In addition,the PCR primers were designed to introduce a standard Kozak sequence(Kozak (1987) Nucleic Acids Res. 15(20): 8125-8148, incorporated byreference herein in its entirety) at the 5′-ends of both the light andheavy chain cDNAs to allow efficient translation, and to introducesplice donor sites at the 3′-ends of both the light and heavy chaincDNAs for the variable regions to be spliced to the constant regions.The PCR primers used for the construction of the chimeric 19/2 light andheavy chains were as follows: catgtttaaacgccgccaccatgggcttcaagatggagtca(5′ end, light chain variable region, SEQ ID NO: 31);agaggatccactcacgtttcagttccacttggtcccag (3′end, SEQ ID NO: 32);catgtttaaacgccgccaccatggagctgatcatgctcttcct (primer for the 5′ end ofthe heavy chain variable region, SEQ ID NO: 33); andagaggatccactcacctgaggagactctgagagtggt (primer for the 3′ end of theheavy chain variable region, SEQ ID NO: 34). The DNA and amino acidsequences of the mouse 19/2 VL and VH regions were adapted for use fromthe construction of chimeric 19/2 light and heavy chains. The entire DNAsequences of mouse 19/2 light and heavy chains cloned into theeukaryotic expression vectors pREN-Neo and pREN-DHFR, respectively, areset forth as SEQ ID NO: 35 & 36, with the resulting light and heavychains resulting in chimerized molecules. Specifically, in SEQ ID NO:35, nucleotides 1357-1756 encode the murine, light chain sequence, withnucleotides 1763-2206 encoding the human kappa region. Within thissequence (1763-2206), a 120 base pair region constituting an intron andsplice acceptor site begins at nucleotide 1886. Within SEQ ID NO: 36,nucleotides 1357-1770 encode the murine 9/2 heavy chain constantsequence with a splice donor site. Nucleotides 1777-2833 encode thehuman IgG1 constant region. Within this sequence, there is a 60 basepair intron region and splice acceptor site which precedes the codingregion.

Example 5

The objective of the experiments described herein was to create stablecell lines expressing chimeric 19/2 (c19/2) anti-human GM-CSF monoclonalantibodies (mAb) in CHO (Chinese hamster ovary) DG44 cells and to testthe secreted antibody for its binding properties. To do this, the DHFRnegative CHO cell line DG044 was used. See Morris et al. (1990) Gene94(2): 289-294; incorporated by reference herein in its entirety). TheCHO cells were cultured in RPMI, supplemented with 10% FCS andHypoxanthine-Thymidine. DNA for transfection was purified from E. colicells using a commercially available product, and the instructionsprovided therein. All DNA preparations were examined by restrictionenzyme digestion. Sequences of chimeric 19/2 mAb variable regions intheir respective vectors were confirmed using an ABI PRISM 310 or LICORSequencer.

Vectors encoding heavy and light chains of chimeric 19/2 mAbs wereco-transfected simultaneously into CHO DG44 cells growing at log phase,using electroporation (270V, 975 μF). Cells were plated in 10 cm dishesand cultured with standard medium. Twenty-four hours later, medium washarvested and replaced by fresh RPMI medium supplemented with 10%dialyzed FCS and 500 μg/mL geneticin. After the initial phase of cellkilling was over (7-10 days), GMP-grade methotrexate was added at aconcentration of 5 nM and gradually increased to 100 nM over thefollowing weeks. Out-growing colonies were picked and screened forantibody production.

Example 6 PCR Amplification of Variable Chain DNA

CHO DG44 cells were centrifuged in an Eppendorf microcentrifuge,briefly, at full speed, washed once with PBS, and pelleted once again.Genomic DNA was prepared by ethanol precipitation after SDS lysis andProteinase K treatment of the cell pellets.

A mixture containing one of the primer pairs described supra, dNTPs,buffer, and Pfu polymerase was used to amplify either the heavy or lightchain variable region using genomic DNA as a template using methods wellknown in the art. The resulting PCR products were digested with theappropriate restriction enzyme and analysed by agarose gelelectrophoresis to confirm their identity.

The primer pairs for the light chain were:

ttcttgaagt ctggtgatgc tgcc, (SEQ ID NO:37) and caagctagcc ctctaagactcctcccctgtt. (SEQ ID NO:38)

For the light chain and SEQ ID NO: 37 plus

gaactcgagt catttacccg gagacaggga gag (SEQ ID NO:39)for the heavy chain.

The undigested heavy chain PCR product had a predicted size of 1200 basepairs, while the light chain PCR product had a predicted size of 800base pairs. Identity was verified by restriction enzyme digest withBamHI.

Example 7 Dot-Blot Method for Measuring Assembled IgG1/Kappa Antibody inCHO Cell Supernatants

CHO cell lines were transfected with the corresponding plasmids.Geneticin resistant cells were obtained and these cells were furtherselected for resistance to methotrexate. Single colonies were pickedafter amplification and transferred into 24-well plates. Culturesupernatant was tested for chimeric IgG 3-4 days later by standard DotBlot assays.

Any positive colonies were sub-cloned and cultured to achieve sufficientantibody production. The chimeric 19/2 antibody was purified from thesupernatant on protein G columns and tested for its specific bindingwith recombinant GM-CSF by Western Blot (FIG. 1) and ELISA (FIG. 2).

Finally, the identity of producer cell lines were confirmed using PCRamplification of both their heavy and light chain variable regions. TheDNA sequence of the heavy chain variable region PCR products forchimeric 19/2 mAb transfected cells was confirmed.

Example 8

In order to optimize cell growth and antibody production, theCHODG44/pREN c19/2 cell line was first cultured in commerciallyavailable IMDM containing 10% FCS, at 37° C., in a 10% CO₂ atmosphere.The cells were then weaned into serum free medium, and cultured in acustom made medium, i.e., IMDM SFII, with the following additives, at37° C., in a 10% CO₂ atmosphere.

Final Concentration Base IMDM Medium Pluronic F68 1.0 mg/ml Hypep 46011.0 mg/ml Hypep 4605 DEV 0.5 mg/ml HEPES 5.958 mg/ml Na₂HCO₃ 3.024 mg/mlAdditives Dextran sulfate 50.0 μg/ml Putrescine 100.0 nM Albumax I 2.0mg/ml Choline chloride 1.0 mg/ml Trace elements FeSO₄.7H₂0 0.8 μg/mlZnSO₄.7H₂0 1.0 μg/ml CuSO₄.5H₂0 0.0025 μg/ml C₆H₅FeO₇.H₂0 5.0 μg/mlIGF-1 50.0 ng/ml Transferrin 35.0 μg/ml Ethanolamine 50.0 μMMercaptoethanol 50.0 μM

Culture supernatants were harvested asceptically, and then clarified bycentrifugation. The antibodies were then purified by affinitychromatography on a 5 ml protein. A Sepharose® fast flow column that hadbeen pre-equilibriated in 50 mM Tris-HCL, pH8, was used. The column waswashed, 20 times, with this buffer, and any bound antibody was elutedusing 50 mM sodium citrate, pH 3.0, and the eluate was then neutralized,immediately, using 1M Tris-HCl, pH8. Antibodies were concentrated with acentrifugal filter, and dialyzed overnight at 4° C. in PBS. The yieldwas about 4-5 mg/liter. The purity of the antibodies was examined viaSDS-PAGE, under both reducing and non-reducing conditions, using a 4-20%gradient on the SDS-PAGE.

Purified antibodies migrated as a single band under non-reducingconditions, and separated into the heavy and light chains, as expected,under reducing conditions.

The antibodies were also analyzed via size exclusion chromatography,(0.5 mg/ml), on a precalibrated HPLC column. Running buffer (5%n-propanol/PBS (0.5 M phosphate, 0/25 M NaCl, pH 7.4)) was used, at aflow rate of 0.2 ml/min at a temperature of 22° C., which is ambientcolumn temperature.

The analysis demonstrated the integrity of the antibodies, which hadcalculated molecular weights of 179 kilodaltons.

Example 9

The experiments described in this example were designed to determine thebinding activity of the antibodies.

Biosensor analyses were carried out using a commercially available,Biacore® 2000, and a carboxymethyldetran coated sensor chip. The chipwas derivatized with 1000, 300, or 100 RVs of recombinant human GM-CSF,on channels 1, 2, and 3 of the machine using standard amine couplingchemistry with channel 4 retained as the control blank channel.

Samples of the chimeric antibody were diluted in HBS buffer (10 mMHEPES, pH 7.4, 150 mM NaCl, 3.4 mM di-NA-EDTA, 0.005% Tween-20®), andaliquots were injected over the sensor chip at a flow rate of 1 μl/min.After injection, dissociation was monitored by allowing NBS buffer toflow over the chip surface for 5 minutes. Any bound antibody was theneluted, and the chip surface was regenerated, between samples, viainjecting 40 μl of 100 mM HCl, pH 2.7, at a rate of 5 μl/min. In orderto carry out kinetic analyses of the binding of the chimeric antibody,varying concentrations, ranging from 1-10 nM, were injected over thechip surface, and both apparent association (“Ka”) and dissociation(“Kd”) rate constants were calculated, using a Langmuir 1:1 bindingmodel, with global and local fitting for calculation of Rmax, using B1Aevaluation V3.1 software.

The results indicated that the chimeric antibody had slightly higheraffinity for rhGM-CSF than the murine antibody. The calculated Ka forthe chimeric antibody was 5.1×10⁵M¹s⁻¹ using 100 RU of GM-CSF. Nodissociation was observed, regardless of analyte concentration,precluding Kd determination and indicating very high affinity.

Global fitting of Rmax, using the software referred to, gave an off rateof Kd=1.9×10⁻⁵s⁻¹ and a high affinity for the chimeric antibody of2.69×10¹⁰M⁻¹.

Example 10

These experiments were designed to determine both the binding activityof the antibodies, and if they cross-reacted with each other.

Nunc plates were coated with recombinant human GM-CSF (1 μg/ml), incarbonate buffer (pH 9.6, 0.05 M), 50 μl/well, and were incubated at 4°C., overnight, and were then blocked with 3% FCS/PBS at roomtemperature, for one hour.

Half-log, serially diluted triplicate 100 μl samples of either murine orchimeric antibody (10 μg/ml) were added to each well, to yield finalconcentrations of from 1.0 ng/ml to 10 μg/ml. Following incubation for 1hour at room temperature, either goat antimouse IgG or antihuman IgG,labelled with horseradish peroxidase (10 ul/well Fc specific; 1:1000dilution in 1% FCS/PBS) were used to detect bound antibody. Afterextensive washings, the bound antibodies were visualized by the additionof ABTS substrate (100 μl/well).

Optical density was read at 415 nm in a microplate reader.

The same protocol for binding antibody to the solid phase was used todetermine if the antibodies competed with each other. As in theexperiments, supra, half-log, serially diluted 100 μl samples, intriplicate, of 10 μg/ml of the murine or chimeric antibody were combinedwith 20 μg/ml of competing antibody, and then 100 ml of the mixture wasadded to the coated ELISA plates. Incubation was as above, andanti-murine or anti-human IgG labelled with horseradish peroxidase wasused, also as described supra.

The results indicated that the antibodies did compete for binding forrecombinant human GM-CSF. A shift in the binding curve was effected byaddition of the excess, competing antibody. This indicated binding to,and competition for, a common epitope.

Example 11

These experiments were designed to test the neutralizing activity of theanti-GM-CSF antibodies. Two human GM-CSF dependent cell lines, i.e.,TF-1 and AML-193 were used. Growth curves were established, in thepresence or absence of 0.5 ng/ml of recombinant human GM-CSF, and viablecell numbers were determined, via Trypan Blue exclusion, on day 0, 1, 2,3, 5 and 7.

In a first bioassay, recombinant human GM-CSF, in amounts ranging from0.0003 ng/ml up to 10 μg/ml, was mixed with anti-human GM-CSFantibodies, at a final concentration of 30 μg/ml, in 96 well, microtitreplates. Either TF-1 or AML-193 cells were added (10³ cells/well), andplates were incubated at 37° C. for 7 days.

After this incubation period, the DNA proliferation marker MTS wasadded, at 20 μl/well. Dye incorporation was measured after 2 hours, bymeasuring light absorbance at A_(490nm).

Increased MTS dye incorporation was observed as the amount of rhGM-CSFin the medium increased. Total growth inhibition of both cell types wasobserved with the chimeric antibody when rhGM-CSF concentration was 0.1ng/ml or less, and there was marked inhibition of cell growth at 0.3-10ng/ml rhGM-CSF.

In contrast, while the murine antibody had a similar effect on AML-193cells, it was less effective on TF-1 cells. These results are seen inFIGS. 3 and 4.

In a second bioassay TF-1 and AML-193 cells were grown in the presenceof 0.5 ng/mL rhGM-CSF and increasing amounts of murine or chimeric 19/2mAbs (0.003-100 μg/mL) were added to the culture media and theneutralizing activity assessed after 7 days culture. Results are shownin FIGS. 5 and 6 for the TF-1 and AML-193 cells, respectively. Inagreement with the initial bioassay, the chimeric 19/2 demonstratedmarked neutralizing activity of GM-CSF stimulated cell growth. A directcorrelation was observed between increasing ch19/2 concentration andGM-CSF neutralizing activity plateaued at 3 μg/mL for both cell lines,with higher concentrations unable to effect a greater reduction in TF-1or AML-193 cell growth. These observations may be due to lower affinityof the murine mAb or steric hindrance at the binding site on GM-CSF.

Example 12

Additional experiments were carried out to produce a chimeric, HRS-3antibody. The murine form of this antibody is described by Hombach, etal, Int. J. Cancer 55:830-836 (1993), incorporated by reference. Themurine antibody binds to CD-30 molecules.

The protocols set forth for production of chimeric, anti GM-CSF antibodyset forth supra were used. Since the antibodies were different, andsequences were known, however, different primers were used. Theseprimers serve to introduce splice sites into the cDNA sequences encodingthe murine heavy chain and light chain variable regions, and are setforth at SEQ ID NOS: 44, 45, 46 & 47, with SEQ ID NOS: 44 & 45 thenucleotide and amino acid sequences of the heavy chain, and 46 & 47comparable sequences for the light chain

The primers were:

(SEQ ID NO:40) gcgccatggc ccaggtgcaa ctgcagcagt ca and (SEQ ID NO:41)cagggatcca ctcacctgag gagacggtga ccgt,and for the light chain:

(SEQ ID NO: 42) agcgccatgg acatcgagct cactcagtct cca and (SEQ ID NO: 43)cagggatcca actcacgtttg atttccagct tggt.

Following amplification, the murine heavy and light chain variableregions were cloned into the pREN Neo and pREN-DHFR sequences, which areset forth at SEQ ID NOS: 48 & 49, respectively. The cloning was possiblebecause the amplification introduced PmeI and BamHI restriction sitesinto SEQ ID NO: 44, at nucleotides 1-7, and the final 6 nucleotides.Comparable sites are found at nucleotides 1340-1348, and 1357-1362 ofSEQ ID NO: 48. Similarly, PmeI and BamHI restriction sites wereintroduced at nucleotides 1-8, and the last 6 nucleotides of SEQ ID NO:47, such that this nucleotide sequence could be cloned into SEQ ID NO:49, at positions 1337-1344, and 1349-1354.

The chimeric HRS-3 antibody was designed to have murine HRS-3 VL and VHregions linked to human kappa and gamma-1 constant regions,respectively. PCR primers were used to modify the 5′- and 3′-sequencesflanking the cDNA sequences coding for the murine HRS-3 VL and VHregions. Modification included the insertion of a NcoI site at the 5′primer end and a splice donor site followed by a BamHI restriction siteat the 3′-end of both the light and heavy chain cDNAs for the variableregions to be spliced to the constant regions. These adapted mouse HRS-3variable regions were then subcloned through the NcoI/BamHI restrictionsites into a prokaryotic vector harboring a 5′PmeI site followed by a 5′Kozak sequence and by a human antibody leader sequence. Sequences werecut from the prokaryotic vector by PmeI/BamHI digest and subcloned intomammalian cell expression vectors already containing the human kappa(pREN-Neo vector) or gamma-1 (pREN-DHFR vector) constant regions,described supra.

Example 13

Once the constructs were established, they were transfected into DGO44cells, as described supra.

Positive colonies were sub-cloned, cultured to achieve sufficientantibody production, after which the antibodies were purified, onprotein G columns via the Fc fragment.

The purified antibodies were analyzed via SDS-PAGE, following Laemmli,Nature 227:680-5 (1970), as modified by Renner, et al, Eur. J. Immunol25:2027-35 (1995), incorporated by reference. Samples from differentstages of purification were diluted, in either reducing or non-reducingbuffer, and were separated on 10-12% polyacrylamide gel viaelectrophoreses followed by standard Coomassic staining.

The results were in accordance with production of a complete, chimericantibody, as evidenced by the banding patterns found in both reducingand non-reducing solutions.

Example 14

The binding capacity of the chimeric HRS-3 antibody was determined viaflow cytometry, in accordance with Renner, et al, supra. In brief, 1×10⁶cells of a target tumor line which expressed CD-30 were washed, twice,in PBS, and then incubated with varying concentration of antibody, at 4°C., for 30 minutes. The cells were then washed, and incubated with asecondary antibody, which was directed to the light chain, conjugated toeither FITC or PE.

The results indicated that there was weak binding from cell culturesupernatant purified from transfected CHO cells, and string binding withpurified antibody. No binding was found when CD-30 negative tumor cellswere used.

Example 15

The antibody dependent cellular toxicity (ADCC), and the complementdependent toxicity of the chimeric HRS-3 antibody were determined usinga europium released assay, as described by Hombach, et al, supra, andRenner, et al, supra.

In brief, for the ADCC assay, peripheral blood lymphocytes were isolatedfrom tow healthy donors, and used at an effector:target ratio of 10:1,with 10,000 europium labelled, CD-30 antigen positive L540CY tumorcells. Antibody was added at varying concentrations (10, 1, 0.1 and 0.01μg/ml), as was a control of 0 μg/ml. The effect was compared to themurine antibody, a bispecific murine anti-CD16/CD30 antibody, and anirrelevant, chimeric IgG1 antibody. A CD30 negative line was also used.Maximum lysis was measured after 0.025% Triton was added, and all assayswere carried out in triplicate.

The results indicated that the chimeric antibody performed better in theADCC than the murine antibody.

In the CDC assays, 10,000 europium labelled cells (100 μg) (L540Y), wereincubated, with 50, 5, 0.5, or 0.05 μg/ml antibody in a 50 μl volume.Freshly isolated complement (50 μl) was added, and the mixture wasincubated for 2 hours, at 37° C. The murine antibody was also tested, aswas an anti CD-16 antibody and a chimeric anti IgG antibody, whichserved as controls, as did a CD-30 negative cell.

As in the ADCC assay the chimeric antibody was superior in terms ofpercent lysis to all other antibodies tested.

Example 16

G250 is an antigen also known as “carbonic anhydrase 9,” or “CA9,” or“MN.” The G250 antigen and the corresponding antibody was described asbeing associated with renal cancer carcinoma by Oosterwijk, et al,PCT/US88/01511. The G250 antibody has also been the subject of severalclinical trials (Oosterwijk, et al., Int. J. Cancer 1986: Oct. 15,38(4):489-494; Divgi, et al., Clin. Cancer Res. 1998: Nov4(11):2729-739.

Zavada, et al, have issued a series of patents in which the G250 antigenis referred to as “MN” or “MN/CAIX.” See, e.g., U.S. Pat. Nos.6,051,226; 6,027,887; 5,995,075, and 5,981,711, all of which areincorporated by reference. These parents provide details on the antigen,and describe various tumors in which it is found, including cervicalcancer, bladder cancer, mammary carcinoma, uterine, cervical, ovarian,and endometrial cancer.

Recently, Ivanov, et al, Am. Journal of Pathology 158(3):905-919 (2001),conducted investigations of CA9 and CA12 on tumor cells, and cell lines.

cDNA sequences for the light and heavy variable regions of a murine G250specific antibody are known, and these include the endogenous antibodyleader sequence. PCR primers were used to modify both the 5′ and 3′regions, in order to introduce restriction sites necessary for theintroduction of the coding sequences to the vectors employed, which wereSEQ ID NOS: 48 & 49, supra. The cDNA sequence which encodes the murineG250 heavy chain variable region is set forth at SEQ ID NO: 50, with theamino acid sequence at SEQ ID NO: 51 and the light chain variableregion, at SEQ ID NO: 52, with amino acid sequence at SEQ ID NO: 53. Thefirst 8 nucleotides in each of SEQ ID NOS 50 & 52 represent a PmeIrestriction site. The first 19 amino acids encoded by the nucleotidesequence represent the leader region, and the first 24 the leadersequence for the light chain. The last 6 nucleotides in each of SEQ IDNOS: 50 & 52 are a BamHI restriction site. The same protocol as was usedfor the HRS-3 chimera was used to splice these variable regions into SEQID NOS: 46 & 47.

To secure the cDNA encoding human TNF, a human leukocyte cDNA librarywas used. The peripheral blood lymphocytes were stimulated with PMA, andthe cDNA for TNF was amplified, using standard methods. Restrictionsites were introduced in the cDNA sequence, so that the cDNA for TNF waspositioned right after the hinge region of the G250 heavy chain. A (Gly)Ser coding sequence linked the two. SEQ ID NOS: 54 & 55 set forth thenucleotide and amino acid sequences of a TNF fragment, and SEQ ID NO:56, a construct wherein the human gamma-1 heavy chain is followed by theTNF coding sequence, right after the IgG1 hinge region.

Within SEQ ID NO: 56, nucleotides 1419-1754 encode a partial, human IgG1constant region, containing the CH1 and hinge domain, preceded by a 60base pair intron region and splice acceptor site. The linker, i.e.,(Gly)₄Ser is encoded by nucleotides 1755-1769. The coding sequence forthe human TNF fragment is set forth at nucleotides 1776-2296.

The resulting constructs were transfected into host cells, as describedsupra, and expressed. Note that SEQ ID NO: 56 contains a variant of theheavy chain vector noted supra, as it contains the human CH1 and hingeregions, followed by the TNF encoding sequence.

Cells were transfected and cultured as described supra for the HRS-3chimera, and amplification was carried out using the primers of SEQ IDNOS: 40-43, described supra. The predicted size of the amplificationproduct was 1100 base pairs, and this was in fact confirmed.

Positive colonies were then sub-cloned and cultured, as described supra.The chimeric G250-TNF fusion proteins were purified using anionexchanged chromatography on DEAE columns, using 5 ml samples, andincreased salt concentrations in the elution buffer (NaCl, 0→0.5 M) (pH8). The purity of the fusion proteins was determined, on SDS-PAGE, underreducing conditions. Two bands, of 45 and 28 kDa, respectively,appeared, consistent with the production of a chimeric fusion protein.

The purity of the chimeric fusion protein was confirmed in a sandwichELISA. In brief, plates were coated with 1:6000 dilutions of affinitypurified, goat anti-human IgG serum, and incubated overnight. They werethen blocked with 2% gelatin. Either cell culture supernatant, orpurified antibody was added, at varying concentrations, and thencontacted with biotinylated goat anti-human TNFα specific serum, at 0.1μg/ml, followed by visualization with a standard streptavidin peroxidasereagent.

The ELISA confirmed the purity of the antibody.

Example 17

FACS was carried out, as described supra for the chimeric HRS-3antibodies, this time using the fusion protein, and G250 positive tumorcells. Two different purification runs were tested, with chimeric G250antibody as a positive control, and an irrelevant chimeric IgG1 antibodyas a negative control.

The results indicated that the chimeric fusion protein bound as well asthe chimeric antibody did. No binding was detected when G250 negativecells were used.

Example 18

These experiments were designed to determine if the fusion proteinsretained the ability of TNF to mediate cell death.

This was accomplished using an MTT assay as described by Renner, et al,Eur. J. Immunol 25:2027-2035 (1995), incorporated by reference, and TNFsensitive (“WEHI-R”) cells. The WEHI cells were seeded at a density of10,000 cells/well. Then, after 18 hours, sterile samples of the fusionprotein, recombinant TNF, chimeric G250 antibody, or a negative control(plain medium), were added, at concentrations of 1.0×10⁵, 1.0×10², 1,1.0×10⁻², 1.0×10⁻⁴, and 1.0×10⁻⁵ ng/ml, and the culture was incubatedfor additional period of from 48-72 hours. Any viable cells weredetected, via standard methods, including Annexin V staining, and flowcytometry. To do this, 1×10⁶ WEHI cells were incubated, overnight, withvarying antibody concentrations, and dye positive cells were counted.The effect of antibody loaded tumor cells in WEHI killing was determinedby pre-staining with commercially available PKH-26GL dye.

The chimeric fusion proteins were found to be as effective asrecombinant TNF in killing cells.

Example 19

It is known that TNF stimulates H₂O₂ release by human leukocytes. Thechimeric fusion proteins were tested for this property.

Granulocytes were isolated from blood samples via standard methods, andwere resuspended in reaction buffer (KRPG=145 mM NaCl, 5 mM Na₂HPO₄, 4.8mM KCl, 0.5 mM CaCl₂, 1.2 mM MgSO₄, 0.2 mM glucose, pH 7.35). This mixwas added plates that had been precoated with fibronectin (1 μg/ml, 2hours, 37° C.) to permit granulocyte adherence. Following this, 10011 ofa dye solution (10 ml KRPG+50 μl A6550+10 μl horseradish-peroxidase)were added and incubated for 15 minutes at 37° C. Granulocytes wereadded, at 30,000 cells per well, and then either buffer (KRPG), PMA (5ng/ml), the chimeric fusion protein (1 μg/ml) plus recombinant humanIFN-γ (100 μ/ml), or the fusion protein plus the recombinant IFN-γ (atthe indicated concentrations), were added. H₂O₂ release was measured for3 hours, using standard methods.

The PMA served as a positive control. The chimeric fusion proteininduced H₂O₂ release significantly higher than antibody alone, and theH₂O₂ release increases even more when IFN-γ was added.

1. A recombinant antibody which specifically binds to renal cellcarcinoma associated antigen G250, wherein said recombinant antibodycomprises a light chain polypeptide of an antibody that specificallybinds to renal cell carcinoma associated antigen G250 and a fusionprotein comprising a portion of a heavy chain polypeptide of saidantibody that specifically binds to renal cell carcinoma associatedantigen G250 adjoined via a linker peptide to a fragment of tumornecrosis factor (TNF) consisting of the amino acid sequence encoded bySEQ ID NO: 56 from nucleotide position 1776 to nucleotide position 2296,wherein said portion consists of the variable domain, the CH1 domain,and the hinge region of said heavy chain polypeptide.
 2. An isolatednucleic acid which encodes the recombinant antibody of claim
 1. 3. Anexpression vector comprising the isolated nucleic acid molecule of claim2 operably linked to a promoter.
 4. The expression vector of claim 3,wherein the heavy chain variable domain of said heavy chain polypeptideis encoded by SEQ ID NO:50.
 5. An isolated recombinant cell comprisingthe isolated nucleic acid molecule of claim
 2. 6. An isolatedrecombinant cell comprising the expression vector of claim
 3. 7. Therecombinant cell of claim 5 wherein said cell is mammalian.
 8. Therecombinant cell of claim 6 wherein said cell is mammalian.
 9. Therecombinant cell of claim 8 wherein said cell is a chinese hamster ovarycell.